Review Isolation of Industrial Important Bioactive

Review

Isolation of Industrial Important Bioactive

Compounds from Microalgae

Vimala Balasubramaniam 1, Rathi Devi-Nair Gunasegavan 1, Suraiami Mustar 1, Lee June Chelyn 2 and Mohd Fairulnizal Md Noh 1

1 Nutrition, Metabolism & Cardiovascular Research Centre, Institute for Medical Research, NIH, Level 3,

Block C7, No.1, Jalan Setia Murni U13, Setia Alam, 40170 Shah Alam, Selangor Malaysia 2 Herbal Medicine Research Centre, Institute for Medical Research, Institute for Medical Research, NIH,

Level 3, Block C7, No.1, Jalan Setia Murni U13, Setia Alam, 40170 Shah Alam, Selangor Malaysia

* Correspondence: [email protected]; [email protected]

Abstract: Microalgae are known as a rich source of bioactive compounds which exhibit different

biological activities. Increased demand for sustainable biomass for production of important

bioactive components with various potential especially therapeutic applications has resulted in

noticeable interest in algae. Utilisation of microalgae in multiple scopes has been growing in various

industries ranging from harnessing renewable energy to exploitation of high-value products. The

focuses of this review are on production and use of value-added components obtained from

microalgae with current and potential application in the pharmaceutical, nutraceutical,

cosmeceutical, energy and agri-food industries, as well as for bioremediation. Moreover, this work

discusses the advantage, potential new beneficial strains, applications, limitations, research gaps

and future prospect of microalgae in industry.

Keywords: microalgae; industry; isolation; bioactive compounds; nutraceuticals; pharmaceutical;

cosmeceutical

1. INTRODUCTION

Microalgae are in the form of unicellular, multicellular, filamentous or siphonaceous, known as

photosynthetic microorganism that can be categorized as eukaryotic and prokaryotic [1]. Microalgae

are also the largest global primary producers consist of approximately 200,000 species [2] with

distinctive nutrient contents as well as bioactive compounds which have a wide spectrum of

commercial applications in various facets of industries including pharmaceuticals, nutraceuticals,

cosmeceuticals, biofuels, biofertilisers, wastewater treatments, feed, and proteomics (Figure 1).

Production of microalgae involves mass cultivation, recovery of biomass and downstream processes

for sustainable yield to cater for food, chemical, feed, biofuel, and high value products. Intrinsic

factors such as temperature, salinity, light and the availability of nutrients affect the chemical

composition of the biomasses. Figure 2, illustrates a few of the cultivation processes of microalgae.

The applications of microalgae in industries concentrated to a few specific species which has

high economic value. The highly-sought genera in the global algae market were dominated by

Spirulina and Chlorella in the form of dried biomass due to various beneficial health effects [3]. Among

the myriad components which were exploited for commercial purpose are fatty acids, carotenoids,

vitamin, minerals, polysaccharides and bioactive compounds. According to the latest analysis, the

global market for microalgae is forecasted to reach USD 3,318 million by 2022 driven mainly by the

demand from pharmaceutical and nutraceutical industries [4] owing to customers’ increasing health

concern, interest on natural alternatives as well as escalating chronic diseases. Likewise, various

microalgae derived compounds reported to exert various skin benefits which currently gaining

attention in many aspects of cosmeceutical production.

Preprints (www.preprints.org) | NOT PEER-REVIEWED | Posted: 25 December 2020 doi:10.20944/preprints202012.0658.v1

© 2020 by the author(s). Distributed under a Creative Commons CC BY license.

https://doi.org/10.20944/preprints202012.0658.v1

http://creativecommons.org/licenses/by/4.0/

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1

Microalgae Biomass

Pharmaceutical

Anticancer

Antiinflammatory

Antiviral

Antifungal

Antibacteria

Neutraceutical/

Food

Food

supplement

Gelling

agent

Food

additives

Bio-colorant

Cosmeceutical

Skin

whitening

Skin

disease

Antioxidant

Moisturising agent

Fuel/Energy

Biodiesel

Bioethanol

Electricity

/Heat

Biogas

Fertilizer

Enhance soil fertility

Plant growth

stimulant

Bio-pesticide

ProteomicsWaste water

treatmentFeed

Nutritional source

Enhance fertility

Enhance immune system

Figure 1. Potential uses of microalgae in various industries.



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Figure 2. Lab scale microalgae cultivation types. A) Raceway photobioreactor B) Indoor annular

column photobioreactor, C) Microalgae culture in bottles with light and oxygen in Scientific

laboratory, D) Open annular photobioreactor, (Courtesy: Dr. Adibi Rahiman Md Nor, University

Malaya).

Advances of new application areas of microalgae in aquaculture and biofuel production have

provided a significant rise of algae demand in the global market. Microalgae are constituted of high

level of lipids. Various research has highlighted the potential of microalgae biomass as a source of

renewable energy, namely biofuel which is imperative to reduce the dependency on fossil fuel [5].

Microalgae have an upper hand in the biofuel production compared to other bioenergy sources (corn,

sugar cane, palm oil and etc.) as they do not require arable land for cultivation, thus eliminating

competition for space and resources with food crops. Some of the key players in the algae industries

were Algae Tec, Pond Biofuels Incorporated, Cyanotech, Kai BioEnergy, Algae Systems and others

[4].

The review aims to summarise the value-added components from microalgae with potential

application in the pharmaceutical, nutraceutical, cosmeceutical, energy and agri-food industries, as

well as for bioremediation along with commercial applications and examples of microalgal

manufacturers as well as commercialized products.

2. MICROALGAE BIOMASS

The mass production of algae biomass is important for various industries [6]. Numerous

methods have been established for microalgae-based products development and down-stream

processes with the advancement in the technologies in this area.

2.1. Biomass Production

A B

B

B

C D



4

The process of biomass production of microalgae encloses several steps such as cultivation,

harvesting and biomass dehydration as shown in Figure 3

Figure 3. The production of microalgae biomass [5,6]

2.1.1 Cultivation

There are two systems developed for the production or culturing of algal biomass; the open

pond and closed photobioreactor (PBR) technologies. Open pond production is categorised into two

systems; natural waters (ponds, lakes and lagoons) [7] and artificial ponds (circular and raceway)

[7,8]. The open pond is a cheaper method of large-scale algal biomass production compared to the

PBR. The PBR however, provide an excellent and controlled closed culture systems for cultivation,

preventing hazard or contamination from moulds, bacteria, protozoa and competition by other

microalgae [9]. It is usually placed outdoor to exploit the free sources of energy from sunlight. There

are three types of PBR categorised into tubular (TPBR), vertical column (VCPBR) and flat-plate (FP-

PBR) [10].

2.1.2 Harvesting

The microalga biomass can be separated from the culture medium or harvested by four means;

biomass aggregation (flocculation and ultrasound), flotation, centrifugation and filtration. In some

cases, combinations of two or more techniques are used to increase effectiveness. Harvesting method

selection depends on several criteria of the microalgae such as the density, size and the desired final

products [11].

(a) Biomass aggregation: In the flocculation technique, microalgae cells are aggregated together to

form a larger particle known as floc, with the addition of flocculants such as multivalent cations and

cationic polymers to the media which help neutralised the cells surface charge [12]. There are two

Cultivation

• Open pond (Natural and Artificial)

• Closed photobioreactor, PBR (Tubular, Vertical column and Flat-plate)

Harvesting

• Biomass aggregation (Flocculation and Ultrasound)

• Flotation

• Centrifugation

• Filtration

Dehydration

• Sun-drying

• Spray-drying

• Freeze-drying or Lyophilisation

MICROALGAE BIOMASS PRODUCTION



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types of flocculating agents; chemical and bio-flocculants. The cheaper and easily available chemical

flocculants that are widely used in industry are iron and aluminium salts [13]. However, toxic effects

can occur when using chemical, causing contamination to the biomass. The bio-flocculants which are

safer, eco-friendly and no pre-treatment needed for further downstream processing are the most-

preferred [19,20]. The criteria used to choose flocculants include nontoxic, low-cost and effective at

low concentration. Among Meanwhile, the common biopolymer bio-flocculants used include acrylic

acid and chitosan [14]. In the ultrasound technique, instigates aggregation is initiated followed by

increased sedimentation to facilitate harvesting of the algal biomass [15]. The benefit of using this

method is that the valuable metabolites are preserved because ultrasonic harvesting does not produce

shear stress on the biomass although used continuously [16].

(b) Flotation: It is a technique intended to float algal cells on the surface of the water, using a micro-

air bubbles disperser without the addition of chemicals [17]. This technique is economically

advantage due to the low operational costs with an easy operating procedure and high harvesting of

biomass [18].

(c) Centrifugation: Centrifugation It is the recovery of algal biomass from the culture media using

centrifuge by gravitational force [19]. This technique is rapid, easy and efficient but the cost can

escalate due to the high energy input and maintenance required [20]. Another disadvantage of this

technique is the internal damage of cells, causing loss of delicate nutrients if a high gravitational force

is used [21].

(d) Filtration: Filtration It is a process to isolate alga biomass from the liquid culture medium by

using a porous membrane with various particle size range [22]. It can be implemented through three

different ways; conventional, microfiltration and ultrafiltration (isolation of metabolites). The

conventional filtration is used to harvest large size microalgae (> 70μm) such as Coelastrum and

Spirulina. Microfiltration and ultrafiltration are used to harvest smaller size microalgae, equal to the

size of the bacteria [23]. Filtration may be relatively slow compared to using centrifugation. However,

processing smaller media volumes require lower cost than centrifugation. But in a large-scales

production, centrifugation can be more economical compared to using filtration due to the cost of

membrane replacement and pumping [30].

2.1.3 Biomass Dehydration

Algae biomass is immediately processed to the following stage after being separated from the

culture medium to prevent spoilage or to extend their shelf-life [22]. Three different types of drying

or dehydration process that are normally used include sun-drying, spray-drying and freeze-drying.

The method chosen is entirely dependent on the desired final products.

(a) Sun-drying: It is the cheapest method available compared to the other two techniques. This

technique is solely based on the solar energy which causes limitations in terms of weather condition,

long drying period and the large drying area needed [24]. Since drying using sunlight is an

uncontrollable process, the problem of overheating may occur, change of texture, colour and taste of

the microalgae [22].

(b) Spray-drying: This technique is to produce dry powder from a fine spray of suspension droplets

which is in continuous contact with hot air in a large vessel. This method has many advantages such

as can be operated continuously, the powder produced is very fine and the rapid drying can maintain

a good quality product [25,26]. This method is usually opted for high-value operations due to its

efficiency, but some algal components such as pigments can be significantly deteriorate and the

operation cost is expensive [22].

(c)Freeze-drying or Lyophilisation: It is widely used at laboratory-scale only to dry microalgae since

large scale production can be very expensive [20]. Freeze-drying is a direct dehydration process of

frozen products using sublimation mechanism. The microalgae are frozen to solidify the material

within before freeze-drying. The moisture content of the microalgae is decreased slowly at low-

temperature, maintaining the solid structure and the quality of the product [27].



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2.2 Extraction of Bioactive Compound

The microalgae are composed of carbohydrates, lipids, proteins, minerals and many other

compounds. To utilise the compounds for different application such as for biofuels/energy and

agricultural use, the algal biomass will be pre-treated to release the stored bioactive compound in the

cells [28]. The cell walls will be lysed to enable all the desired components to be extracted and this

may be accomplished by various ways, such as physical, mechanical (bead milling, homogenisation,

microwave, ultrasonic and pulsed electric field), chemical (solvent, acid and alkali) and biological

(enzymes) methods. The method chosen for the pre-treatment process is based on the desired final

products [29,30].

3. MICROALGAE IN PHARMACEUTICAL

Microalgae are potential source for bioactive components with pharmaceutical applications. Several

important microalgae-derived components with their pharmaceutical applications are highlighted

in Table 1.



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Table 1. Important micro-algae derived components in pharmaceutical applications

Compound Microalgae species Isolation/

Extraction method

Cell/ virus/animal model/ clinical

patient

Effect References

Anticancer agents

Lutein Chlorella vulgaris Ethanol extraction and

partitioned with hexane

Colon cancer (HCT116) cells Antiproliferative effect

IC50=40.31±4.43 µg/mL

[31]

Violaxanthin Dunaliella tertiolecta Dichloromethane extract Human mammary carcinoma cell lines

(MCF-7) and human prostatic carcinoma (LNCaP) cells

Potent inhibition of MCF-7 and

LNCaP cells at growth inhibition (GI50) of 56.1 and 60.9 µg/mL,

respectively

[32]

Fucoxanthin Phaeodactylum tricornutum UTEX 640

Pressurized liquid extraction

Human liver cancer cell lines (Hep-G2), Human colon cancer (Caco-2) cells and

HeLa cell line

An inhibitory effect of up to 58% was measured in Hep-G2 cells.

In HeLa and Caco-2 cells, the effect was stronger than that of the

positive control with a final

concentration of 5% DMSO.

[33]

Phycocyanin Spirulina platensis Freeze-thawing followed

by solvent extraction

Liver cancer (Hep-G2) cancer cells, Non-

small cell lung cancer (NSCLC)

Inhibited liver cancer cells and

leukemia cells.

Induce apoptosis in H460 cells

reaching 3.72±0.98% and suppress

growth of NSCLC cells

[34-37]

Phycocyanin Spirulina platensis Supercritical fluid extraction

Human lung cancer cells (A549) IC50=26.82 µg/mL [38]

Phycocyanin Limnothrix sp

37-2-1

Fractional precipitation and

purification with activated charcoal and chitosan

Prostate Cell Line LNCaP C-PC alone (250 and 500 µg/mL)

killed 65% and 70% of cells while C-PC (500 µg/mL) combined with

Topetencan (TPT) (1 µM) killed 80% of cells due to additive effect

[39]

Phycocyanin Limnothrix sp.

NS01

Four-step purification procedure including the

adsorption of impurities

with chitosan, activated charcoal, ammonium

sulfate precipitation, and

ion-exchange chromatography

Human breast cancer cell line (MCF-7) IC50 for 24, 48 and 72 hr exposure to C-PC were 5.92, 5.66 and 4.52

µg/µL

[40]



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Table 1. ( Continued)

Compound Microalgae species Isolation/

Extraction method

Cell/ virus/animal model/ clinical

patient

Effect References

Cardioprotective agents

cis β-carotene

Dunaliella bardawil

(containing a mixture of cis and trans-isomers)

Enriched extract Mice fed a high-fat diet In old mice with established

atherosclerotic lesion, Dunaliella

inhibited significantly plasma cholesterol elevation and atherosclerosis progression

[41]

Eicosapentanoic acid (EPA) Nannochloropsis Enriched EPA oil Healthy subjects Lower cholesterol levels [42]

Antiviral agent

Cyanovirin Nostoc ellpsosporum Aqueous extraction HIV-1 laboratory strains;

HIV-1 primary isolates;

HIV-2; SIV;

HIV-1 laboratory strains (EC50 0.1-

5.8 nM), HIV-1 primary isolates

(EC50 1.5-36.8 nM), HIV-2 (EC50 2.3-7.6 nM), SIV (EC50 11 nM)

[43]

Scytovirin Scytonema varium Aqueous extraction HIV-1 laboratory strain (HIV-1RF); HIV-1 primary isolates (ROJO) in

peripheral blood mononuclear cell

(PBMC); HIV-1 primary isolates (Ba-L and ADA)

in macrophages

HIV-1RF (EC50 0.3 nM), HIV-1 primary isolates (ROJO) in

PBMC (EC50 7 nM), HIV-1 primary

isolates (Ba-L) (EC50 22 nM), HIV-1 primary isolates (ADA) (EC50 17

nM)

[44]

Mixture of Icthypeptins A and Icthypeptins

Microcystic ichthyoblabe Methanol extract Influenza A IC50 12.5 µg/mL [45]

Calcium spirulan Spirulina platensis Aqueous extraction HIV-1;

Herpes simplex virus type 1 (HSV-1)

HIV-1 (IC50 9.3 µg/mL),

Herpes simplex virus type 1 (HSV-1) (IC50 9.0 µg/mL).

[46]

Sulfated exopolysaccharide Porphyridium cruentum Aqueous extraction Herpes simplex virus type 1 (HSV-1); Herpes simplex virus type 2 (HSV-2);

Vericella virus (VZV)

HSV-1 (CPE50 protection 1 µg/mL), HSV-2 (CPE50 protection 5 µg/mL), VZV (CPE50 protection 0.7 µg/mL).

[47]



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Many studies have documented the health benefits of microalgae compounds for prevention and improvement of diseases such as diabetes, obesity,

cardiovascular disease, cancer, inflammation, Alzheimer’s diseases, depression as well as bacterial, fungal and viral infections [42,48,49]. Despite that, only a

limited number of microalgae with pharmaceutical applications are currently available as listed in Table 2. Low extraction yield and high production cost are some

of the factors in delaying commercialization of some microalgae-derived bioactives [50]. In addition, there are potential risk of severe side effects, allergic

reactions and accumulation of heavy metals and toxins in some species of microalgae [51]. Consequently, strong emphasis on the good manufacturing

practices in cultivation, harvesting, extracting and purification and controls to limit toxin and impurities are required to ensure safety, efficacy and quality of

microalgae-derived purified compounds and enriched extracts for approval and commercialization [52].

Table 2. Examples of bioactive components from microalgae that are produced in commercial scale

Microalgae Bioactive Product Pharmaceutical

applications

References

Haematococcus pluvialis

Astaxanthin

Spirulina (Earth Spirulina Group, ES Co, Seoul, Korea)

Lipid lowering [53]

Schizochytrium limacinum Docosahexaenoic acid (DHA) Maris DHA oil (IOI, Hamburg,Germany) Rheumatoid arthritis [54]

Nannochloropsis Eicosapentaenoic acid (EPA)

Almega®PL (iWi Life) (Qualitas Health, Houston, US)

Cholesterol lowering

[42]

Arthrospira FEM-101 Allophycocyanin ApoX surface antiviral spray (FEBICO, Taiwan) Antiviral [55]



10



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3.1 Compounds with anti-cancer properties

Microalgae-derived bioactives with anti-cancer properties are extensively studied in recent years

[33]. Among the compounds, the microalgae pigments such as astaxanthin, β-carotene, lutein,

violaxanthin and fucoxanthin have most potential to be commercialized as pharmaceuticals because

of their established applications as nutraceuticals and cosmetics and the rising demand as dietary

supplements. In fact, astaxanthin and β-carotene are currently produced commercially from

microalgae Dunaliella salina (D. salina) and Hamatococcus pluvialis, respectively while commercial

production of other carotenoids such as lutein and fucoxanthin are gaining momentum [56].

Fucoxanthin for instance, have been isolated from diatom microalgae Phaeodactylum tricornutum

which is cultivated in a pilot-scale photobioreactor and can be considered as a commercially viable

source for fucoxanthin [51]. The anti-cancer properties of some of these carotenoids are summarized

in Table 1. Extraction of carotenoid is achieved using ultra-sound extraction and freeze-thawing

methods however organic solvent extraction at high temperature and pressure are more widely used

method in commercial- scale production [56].

Another promising anticancer agent is phycocyanin, a protein pigment from phycobiliprotein

group. Phycocyanins are isolated from the commercially grown microalgae Spirulina platensis but

isolation from other cyanobacteria such as Limnothrix sp. has also been reported (Table 1) [41,43]. C-

phycocyanin showed inhibitory activity in liver cancer cell lines (HepG2) [57], human leukemia cells

(K562) [58] and against lung cancer cell lines (A549 and NSCLC) [39,40,41]. In another study, the

phycocyanin from Limnothrix sp. enhanced the anticancer properties of the anticancer drug Topetecan

against prostate cancer cell line (LNCap) [42]. Meanwhile, phycocyanin isolated from as Limnothrix

sp. NS01 with 2 subunit α and β (17 and 20 kDa) showed antiproliferative activity in human breast

cancer cell lines (MCF-7) [43]. Isolation of C-phycocyanin from Spirulina platensis was accomplished

using buffer ammonium sulfate solution (i.e. salting- out technique) as well as by using supercritical

fluid extraction using ethanol as a modifier which resulted in higher yield compared to conventional

solvent extraction [41,57]. In the studies, impurities were removed using chitosan and activated

charcoal however further purified the compound by ion-exchange chromatography was also carried

out [43].

3.2 Compounds with cardioprotective properties

Several microalgae-derived compounds have also been studied for its cardioprotective effect

and briefly summarized in Table 1. Carotenoids are shown to possess antioxidant properties that are

important for preventing cell damage caused by free radicals associated with chronic cardiovascular

diseases and stroke [59]. In this regard, some microalgae producing high amount of carotenoids have

been investigated for their cardioprotective properties. For instance, D. salina microalgae can produce

up to 10-13% of β-carotene have been shown to have protective effect against atherosclerosis in both

mouse and humans. Furthermore, a mixture of tran-isomers (~40%) and cis β-carotene isomers (~60%)

from D. salina was found to be more potent in decreasing total lipid, cholesterol and triglyceride (TG)

levels compared to all trans-β-carotene found in synthetic β-carotene [60]. Harari et al. [44] also

showed that cis β-carotene isomers (~50%) from Dunaliella bardawil powder inhibited atherosclerosis

progression in older mice with a high-fat diet. Although cis β-carotene is still not produced

commercially due to its high production costs, a high density inoculum enriched with cis β-carotene

strain from D. salina are currently being developed to deliver reproducible, low-cost D. salina biomass

containing high content of 9-cis β-carotene [31].

Another group of bioactive with cardioprotective properties are the polyunsaturated fatty acids

(PUFAs) especially the omega-3 fatty acids such as DHA, EPA and α-linoleic acid (ALA) which have

been shown to reduce blood cholesterol and improve hypertension. Of these fatty acids, DHA is the

only PUFA currently commercially available. Purified EPA sourced from various microalgae

including Porphyridium purpureum and Isochrysis galbana are still not economically competitive to be

produced commercially. However, a proprietary strain of microalgae Nannochloropsis cultivated in



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an open pond with high solar radiation was shown to produce high EPA content (>65%) oil marketed

as A2 EPA PureTM for supplement and pharmaceutical applications have been reported [61]. PUFAs

are commercially extracted using hexane solvent followed by mechanical pressing. However,

extracted PUFAs are prone to oxidation and therefore all materials that can initiate oxidation such as

copper are eliminated from the extraction and storage area [61].

3.3 Compounds with antiviral properties

Microalgae are also potential sources for bioactives with antiviral properties (Table2). For

example, lectin protein with antiviral properties such as cyanovirin (CV-N) and scytovirin (SVN)

have been reported [46,47]. CV-N is a 11kDa protein consisting of 101-amino acid in single chain with

two disulfide linkages and was isolated from the aqueous extract of Nostoc ellpsosporum and purified

by ethanol precipitation followed by fractionation and purification by column chromatography [62].

CV-N showed a broad spectrum of antiviral activity against human immunodeficiency virus-1 (HIV

type-1) laboratory and clinical strains with effective concentrations (EC50) range 0.1-5.8 nM and 1.5-

36.8 nM, respectively [46]. In the same study, CV-N was also active against human immunodeficiency

virus-2 (HIV-2 ) and simian immunodeficiency virus with EC50 2.3-7.6 nM and 11 nM, respectively

[46]. Although CV-N showed promising antiviral activity including in an animal HIV transmission

model the clinical application of this molecule is currently limited due to its reported mitogenic

activity [63]. Meanwhile, another lectin protein SVN has been isolated from the aqueous extracts of

cultured cyanobacterium Scytonema varium. SVN is a 9.71kDa protein consisting of 95-amino acid

chains with 5-disulfide linkage which also showed potent antiviral activity against HIV type-1

laboratory strains and clinical isolates with EC50 between 0.3-22 nM [47].

Another group of compounds with antiviral activities are the cyclic peptides. The fractions

containing a mixture of Icthypeptins A and Icthypeptins B, cyclic depsipeptides isolated from

Microcystic ichthyoblabe showed antiviral activity against influenza A virus with inhibitory

concentration (IC50) of 12.5µg/mL comparable to the control amantadine IC50 of 15µg/mL [64]. Besides

that, sulfated polysaccharides with antiviral properties have also been described. For example,

calcium spirulan isolated from Spirulina platensis showed antiviral activity against HIV-1 with IC50 of

9.3 µg/ml comparable to the dextran sulfate control when assessed by P24 antigen assay [49]. The

sulfated exopolysaccharide (1 µg/mL) from the Spanish strain of Porphyridium cruentum showed

strong inhibitory effect on the cytopathic effect on Herpes simplex virus-1 (HSV-1), Herpes simplex

virus-2 (HSV-2) and Vericella virus (VZV) with CPE50 protection of 0.7-5 µg/mL [50].

4. MICROALGAE IN NUTRACEUTICALS/FOOD

A number of microalgae have been classified as Generally Regarded as Safe (GRAS) and

approved by US Food and Drug Administration (FDA). For the guaranteed safety and valuable

source of nutrient, algae are used widely in industries especially for food and nutraceutical

applications. According to Watanabe [65], microalgae species of cyanobacteria eg. Spirulina,

Aphanizomenon and Nostoc are hugely harvested for the food industry. Dried Aphanizomenon,

contributes approximately 500 tons annually which is dominantly produced in North America at

Upper Klamath Lake, Klamath Falls, Oregon for food supplement industry while Spirulina is widely

cultured and produced in countries like United States, Taiwan, China, India and others with an

estimated output of 3000 tons annually [66]. Myanmar Spirulina factory in Yangon produces tablets,

chips, pasta and liquid extract [3]. Other species like Chlorella sp, Haematococcus sp, Dunaliella sp.

cultivated commercially by various countries for nutraceuticals and food application. List of

commercialized strain, industry application, companies and the countries involved is provided in

Table 3.



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Table 3. List of commercialized strain, industry application, companies and the countries involved

Biomass

Extracted compound Isolation/Extraction method Application in industry Company Country References

Spirulina platensis

Algae protein; Biomass

Enzymatic hydrolysis, Physical processes, Chemical extraction,

Ultrasound-assisted extraction,

Pulsed electric field, and Microwave-assisted extraction

Health supplement, Health food, Infant

formula

Saxony-Anhalt; Parry Nutraceuticals;

Japan Spirulina Co., Ltd;

Siam Alga Co., Ltd.

Germany; India;

Japan;

Thailand

[3,67,68]

Spirulina platensis Vitamin 12 Water extraction method Health supplement Myanmar Spirulina Factory Myanmar [68,69]

Chlorella vulgaris

Chorella sp.

Biomass, pigments Microwave-assisted extraction Health food, food

supplement

Nikken Sohonsha Corp.;

Chlorella manufacturing and

Co.; Klötze;Ocean Nutrition

Japan;

Taiwan; Germany;

Canada

[3,68]

Haematococcus pluvialis Astaxanthin Mechanical treatments, chemical

treatments using solvents, pressurized extraction,

ultrasounds and microwaves

Food supplement, bio-

colorant

Algae Health Science;

Cyanotech Corporation; Aquasearch

Algatechnologies

China;

US; Israel

[68,70-73]

Dunaliella salina Beta-carotene Solvent extraction; Microwave-

assisted extraction

Health food, Dietary

supplement, bio-colourant

Cyanotech;

Earthrise Nutritionals;

Nature Beta Technologies

Cognis; Betadene;

Cognis Nutrition and Health

US;

Australia

[68,74]

Spirulina platensis Phycocyanin Microwave-assisted extraction Bio-colourant, Health supplement

Panmol/Madaus;

Yunnan Green A Biological

Project Co., Ltd

Austria;

China

[68]

Ulkenia sp. DHA Solvent extraction and microwave Dietary supplement; Nutrinova German [3]

Schizochytrium sp. DHA Cellular hydrolysis Dietary supplement; Health food

OmegaTech USA [3]



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Table 3. (Continued)

Biomass

Extracted compound Isolation/Extraction

method

Application in industry Company Country References

Crypthecodinium cohnii DHA Microwave Infant formula Martek USA [75]

Nannochloropsis oculata Omega-3 PUFA Solvent extraction; Microwave

Omega-3 supplements Qualitas;

Cleanalgae SL; Astaxa

USA;

Spain; Germany

[76,77]

Porphyridium spp Polysaccharides Ultrafiltration Food additives, Nutrition

InnovalG France [68,78,79]

Euglena gracilis Biomass Flocculation Health food Euglena Japan [80]

Odontella aurita Fatty acids Health supplement InnovalG France [68]



15

54.1 Algal Protein

Demand for plant-based nutrient especially protein sources has augmented over the years owing

to growing health concern and shift of millennial preference across the world to nutraceutical

products which are convenient and offers a high value of nutrition. This, in turn, has the

manufacturers and industries of food and beverages as well as nutraceuticals to search for lucrative

sources of protein. Among all, microalgae showed as a promising source of protein combined with

diverse bioactive compounds and essential nutrients. The demand for global algae protein exceeded

USD700 million in 2019 and projected to expand over the years in view of changing lifestyle of

consumers and preference [81].

Among the myriad of algae, Chlorella and Spirulina species are most sought-after in the global

microalgae market owing to its high protein content (50%-70% protein of its dry weight) and broad

spectrum of other nutrients viz. minerals, vitamins, lipids, carbohydrates, pigments and other trace

elements [82]. Tavelmout Corp., a biotech company based in Japan developed a closed flat panel

photobioreactor system which enhances protein productivity in Spirulina about 20 times higher than

that of soybeans [83]. While, Siva Kiran et al. [84] reported that the protein content in Spirulina is

higher compared to other foods such as milk, chicken, beef, and some plants. In food application,

Spirulina and Chlorella or its protein were incorporated in various types of food, such as milk-based

products, bread, biscuits, instant noodles and pasta to produce protein-enriched functional food [85-

88].

The extraction of protein from microalgae comprised steps including cell disruption, extraction

and product purification; cell disruption techniques involve mechanical action (high-pressure

homogenisers, bead mills), ultrasounds, enzymatic or chemical treatments, thermal or osmotic shocks

(repeated freezing/thawing) [89]. For efficient protein recovery (76%) from Chlorella sp., Ursu et al.

[90] suggested an alkaline treatment followed by isoelectric precipitation. Meanwhile, Chia et al. [91]

proposed an effective approach using ultrasound-assisted three phase partitioning method for

efficient protein extraction and an optimised conditions for high protein recovery which is applicable

for the future integrated bio-separation technique for biomolecules extraction from microalgae as

well as to improve the current downstream bioprocessing techniques.

Chlorella and Spirulina are known for its high-quality protein attributed by the well-balanced

amino acid composition according to FAO/WHO recommendation [92], digestibility coefficient, as

well as by its biological value of the amino acids absorbed from the food [93]. Nevertheless, interest

to acquire good quality protein for human nutrition continues with exploration on different strains

of microalgae, as such, a study on Australian microalgae species in James Cook University/MBD

Energy Research facility using Scenedesmus sp., Nannochloropsis sp., Dunaliella sp., and a designed

freshwater chlorophytic polyculture (CPC; consisting of Schroederiella apiculata, Scenedesmus

pectinatus, Tetraedrom minimum, Mesotaenium sp. and Desmodesmus sp.) exhibited high quality protein

in all studied microalgae. The protein was suitable for human consumption which was determined

and supported by Essential amino acid index (EAAI) and was comparable to the commercial Spirulina

and Chlorella products. The Australian strains earned higher score for EAAI due to presence of higher

essential amino acids such as histidine, phenylalanine, threonine and lysine. In addition, the selected

strains also displayed a comparable nutrient strength and taste as the Spirulina and Chlorella species

respectively, thus, suggesting the potential of these microalgae for future commercialization in

human nutrition area [94].

54.2 Vitamins and minerals

Microalgae constitute important source of almost all vitamin and essential minerals. Spirulina

was reported as rich source of vitamins B1, B2, B12 and high content of amino acid up to 62%, [95], all

of which have facilitated its claim as superior to other microalgae [96]. Seghiri et al. [95] also suggested

that Spirulina benefits may be attributed by the presence of macro-minerals as well as the trace

elements. Various studies have shown that Spirulina as a good source of pro-Vitamin A, in term of



16

beta-carotene and exhibited better effects than the synthetic Vitamin A due to its good bioavailability

[97,98]. This species also was reported for high content of Vitamin B12, in the range of 120-244 µg per

g dry weight, however, studies suggested that most of it were pseudo B12, an analogue which has a

similar structure and bound to specific B12 transporter but does not exert any health benefit [65,99].

In contrast, the latest study by Madhubalaji et al. [100], provided a scientific validation for the use of

Spirulina as Vitamin B12 source. However, more studies needed to substantiate Spirulina as a viable

source of Vitamin B12 in human. In view of high bioavailability of iron from Spirulina, Puyfoulhoux

et al. [101] concluded that Spirulina constitute adequate source of iron for human consumption.

Chlorella is another commercial species with a rich source of proteins, essential amino acids,

vitamins (B‐complex, ascorbic acid), minerals (potassium, sodium, magnesium, iron, and calcium).

Chlorella sp. is suggested to be the best candidate for vegan source of Vitamin B12 and used in food

supplement [102]. According to Kumudha et al. [69], Vitamin B12 detected in the Chlorella vulgaris

present as methylcobalamin, a biologically active form suitable for human consumption while

Merchant et al. [103] reported that participant supplemented with 9 g Chlorella pyrenoidosa daily

mitigated vitamin B12 deficiency in vegetarian and vegan participants. A study by Nakano et al. [104],

suggested 6g Chlorella supplementation significantly reduced the risk of pregnancy-associated

anaemia, proteinuria and oedema. All of these, suggest microalgae enriched food as a good source of

nutritional supplements especially for strict vegetarians owing to its rich high-value nutrients as well

as Vitamin B12. Chronopoulou et al. [105] proposed an improved approach to extract fat-soluble

vitamins by using supercritical CO2 while a previous study compared six extraction methods for

Vitamin B12 from Spirulina and found the aqueous extraction suits best for this studied compound

[106].

4.3 Fatty acids

Microalgae contain distinctive profile of lipids especially the fatty acids, for example, EPA or

DHA with feasible commercial value. The ability to produce and accumulate high amount of PUFAs,

makes microalgae even more valuable as nutraceutical. Omega-3 fatty acids known as a good source

of dietary supplements which highly recognised and recommended for its health benefits specifically

in disease prevention [107] and human nutrition. The algal oil often used in liquid or capsule form

and benefits in particular, vegetarians as well as populations with low seafood diets. Application of

algal oils in food achieved popularity after advancement in the microencapsulation and refining

technology which prevents any off-flavour towards the food when combined together with the oils.

The algal oils are enriched or fortified in myriad of foods such as dairy products, nutritional bars,

bakery products and etc. [108] to further increase product nutritional value.

Microalgae produce total lipid up to 30-70% of dry weight depending on the species; which

plays a critical role as energy stockpile during adverse condition and cell division. Fatty acids are

component of complex lipids and the structural variation attributes to their multitudinous benefits.

Based on the polarity of the molecular head, fatty acids can be categorised into two groups; i) neutral

lipids and ii) polar lipids. Fatty acids production can be increased in the microalgae by manipulating

various environmental factors such as oxygen level, temperature, light exposure, pH as well as

limiting the nutrient supplementation [109]. A number of studies have suggested nitrogen starvation

to increase the neutral lipid and triacylglycerides (TAG) synthesis in microalgae [110,111].

Meanwhile, exposure to low light intensity and therefore low temperature was reported to enhance

PUFAs production [112,113]. Chlorella sp. culture in low CO2 showed to promote high contents alpha-

linolenate fatty acid, in contrast Chlamydomonas reinhardtii mutant cia-3 exhibited higher content of

PUFAs in culture condition with high CO2 concentration [114,115].

Methods of harvesting lipid from microalgae include mechanical pressing, homogenization,

milling and solvent extraction. The solvent extraction is imperative to the polarity or/and solubility

of the lipid of interest. Other techniques such as enzymatic extraction, supercritical extraction,

ultrasonic-assisted extraction, microwaves are used to facilitate lipid extraction by solvent [116].

Nevertheless, only a few cell disruption techniques feasible for commercial application, namely,



17

steam explosion [117], enzymatic hydrolysis, bead milling and horn sonication [116]. Purification of

algal oil (PUFAs) is an essential step as the crude form of these oils are inedible due to its impurities,

odour, taste and unattractive for consumption as its turbid appearance. Speed of operation and

process condition are critical factors in the oil purification techniques as the oil is sensitive to

oxidation. Manufacturer such as Martek Bioscience Corporation has described the oil recovery and

purification steps of DHA from algae oil. For instance, protease enzyme was used to break the protein

in the cells of Schizochytrium sp. to release the oil into the culture broth forming an emulsion, thereafter

isoprophyl alcohol was added to separate the oil. While for Crypthecodinium cohnii, hexane solvent

extraction method was applied since the enzyme method is incapable to hydrolyse the algae cellulosic

layer. Following the solvent extraction, the cell walls are removed by centrifugation and the oil was

recovered after solvent evaporation [118].

Several studies and reviews have been published on the different algae strains, namely,

Nannochloropis oculata [76,77], C. cohnii, Schizochytrium sp., Ulkenia sp. which are associated with the

presence of high concentration of PUFAs in its lipid [119] and manifested to have better

bioavailability compared to other fish oils [77]. These strains have been commercially used to

develop high purity marine oils by companies such as Qualitas Health, DSM-NP, Lonza and GCI

Nutrients. DHA oil from C. cohnii (40-50%) are fortified in infant formula milk by company such as

Martek, USA, thus, the cultivation and manufacturing processes follow a strict regulation of FDA

and current Good Manufacturing Practice (cGMP). This fortified formula sold in more than 60

countries [3]. Recently, two more strains with potential commercial values namely, Phaeodactylum

tricornutum and Porphyridium purpureum for EPA and other compounds were suggested [120,121].

4.4. Natural pigments

Microalgae are also a rich source of pigments which are used as bio-colourant or food additives

in various products. Natural pigments are highly coveted by food industries as an alternative to

synthetic sources which has various health implication [122]. One of the key players in this natural

pigment industry is a Chinese astaxanthin supplier, Algae Health Science who operates one of the

biggest facilities in the world, using glass tubes to cultivate Haematococcus pluvialis microalgae for

astaxanthin extraction in Yunnan, China (Figure 4) [123].

Figure 4. Closed tube bioreactor algae production facility in Yunnan Province, China (Courtesy:

Nicholas Cheong, Nova Laboratories Sdn. Bhd.).

Closed system cultivation was favoured by this company as it provides better purity and

prevents contamination, independent of local weather as well as providing consistent production.



18

Astaxanthin from Haematococcus has been marketed under various name and companies, as such

Cyanotech Corp. from the US commercialized astaxanthin under the name of BioAstin [124],

AstaReal by Fuji Chemical Industry, Japan, Astaxanthin GoldTM by Nutrigold as well as various

patent applications for dietary supplements, health supplements, as antioxidanst, beverage

colourants and other functions [125,126].

Chlorophylls, a photosynthetic green pigment present in microalgae as chlorophyll a (blue-green

colour), b (brilliant green), c (yellow-green), d (brilliant/forest green) and f (emerald green) dependent

on types of algae [127]. Chlorophyll a, which is abundantly found in cyanobacteria such as Chlorella,

has been extensively used as colouring agent due to its stability [128]. Another prominent colourant

from the microalgae with commercial applications is phycobiliproteins, a water-soluble fluorescent

pigment commonly present in cyanobacteria which can be classified as four major groups according

to their colours and light absorption characteristics, namely, phycoerythrin, phycocyanin,

allophycocyanin and phycoerythrocyanin [129,130]. The phycobiliproteins were shown as a strong

antioxidant which contributes to high- value nutraceutical product and its application in food mainly

in dairy products, chewing gums, candy, beverage mixes and ready-to-eat cereals [131,132]. Besides

being a functional ingredient and safe food colourant, this protein (eg. deep blue colour protein C-

phycocyanin) are used as dietary supplement coating and functional food additives [88].

Since the global demand of natural colourant especially astaxanthin is growing at an

unprecedented rate mainly for food supplements, thus, their average prices in the market are in the

range of 2,500 USD per kilograms and its annual worldwide worth of 200 million USD [133]. Species

like Dunaliella ascribed to its high beta-carotene contents, contributes a total turnover of about 75

million USD where 60 million from it contributed by food supplements [67].

Many reviews and research papers offer detailed steps on how to improve and enhance related

algae cultivation and extraction methods. Development of spiral-tube photobioreactor to cultivate D.

salina contributes to an enhanced and continuous cultivation for high output of β-carotene [134].

Additionally, factors such as high light intensity, temperatures, nutrient limitation and high salt

concentrations increase the β-carotene production [135]. A recent study by Xu and Harvey [136],

suggested the use of high-intensity red light with sufficient nutrient content for high carotenoid

production by up-regulating the whole biosynthesis pathway of carotenoids. Besides its applications

as a food colourant, beta-carotene is used as an additive in multivitamin supplement and tablets [3].

Harvesting and extraction of certain algal strain can be challenging due to its nature, as such D.

salina harvesting deemed to be most difficult and costly compared to other commercial strains

attributed to its lacking of cell walls, low concentration and small size. Many approved techniques

have been developed to tackle the problem such as centrifugation and flocculation, a patented

method involving mechanical harvesting [137] while Pirwitz et al. [138] suggested centrifugation

without flocculation method which was identified as most cost-effective technique. Other commonly

used extraction methods for pigments such as astaxanthin are by mechanical treatments, chemical

treatments, pressurized extraction, ultrasound and microwaves among others while a few researches

have taken a greener option by using non or less toxic solvents such as acetone and ethanol for the

extraction process [71]. Molino et al. [70] suggested a mechanical pre-treatment before accelerated

extraction using green solvents.

A continuous search for potential new strains for large- scale production of these pigments are

still advancing as the current major strains are still limited to a few species, namely, Spirulina

platensis, Haematococcus pluvialis and Chlorella sp. Singh et al. [139] discovered Asterarcys quadricellulare

PUMCC 5.1.1 strain, a green microalga with a promising characteristic for carotenoid production

while Kaushal et al. [140] reported a new cyanobacterium Nodularia sphaerocarpa PUPCCC 420.1 which

produces phycobiliprotein and reckoned as a good candidate for production at commercial scale.

5. MICROALGAE IN COSMECEUTICALS



19

Skin entity is the largest organ which acts as a physical barrier to protect the human body from

harmful external agents, and is composed of three main layers (epidermis, dermis, hypodermis) [141-

143]. A healthy and radiant skin is maintained via a balance between synthesis and degradation of

the matrix proteins such as collagen, elastin and glycosaminoglycans (hyaluronic acid). The

corresponding proteinases coupled with continual synthesis are the maintenance mechanism

involved in this process. However, this balance is affected via both chronological as well as photo-

ageing by coupling the proteinases up-regulation, where this enhances protein degradation along

with synthesis down-regulation. Protein synthesis simulation or alteration of their breakdown via

proteinases is the suggested mechanism of action for improving this inequality [144].

The synthesis of matrix proteins or proteinases inhibition are attempted with the application of

a wide range of specific skin formulation products containing compounds such as ethanolamines,

sodium lauryl sulphates, polypeptides or oligopeptides. However, the possibility of developing

allergic reactions and other adverse conditions led to continuous search for natural bioactive

compounds that could be applied in cosmetic formulations. As such, bioactive compounds

originating from microalgae have gained much popularity of its amazing moisturizing, thickening,

pigmenting, anti-ageing, skin whitening, sunscreen protection, and many other properties [143,144].

In a review by Mourelle and colleagues, cosmetics are defined as products aimed for improving

skin appearance, structure and morphology in assistance of excipients and active ingredients

specifically suited for various skin types [145]. Several microalgae species extracts are widely applied

in cosmetic-based industries, especially for skincare products [146]. These include face and skincare

(anti-ageing cream, emollient, anti-irritant in peelers, refreshing care, sunscreens) as well as hair care

products [146]. Among the common microalgae species include Arthrospira sp., C. vulgaris, D. salina,

S. platensis, Chondrus crispus, Mastocarpus stellatus, Ascophyllum nodosum, Alaria esculenta and N. oculate

[147]. In cosmetic industry, Cu-Chl (CI 75810) is applied for use in hair colour products, colour

cosmetics and bleaching products and categorised as non-toxic [143]. Figure 5 highlights the main

potential applications of various microalgae in cosmetic industry, accounted by its metabolites or

bioactive compounds, which are described in detail below.

Figure 5. Potential applications of microalgae in cosmeceuticals.



20

5.1. Anti-ageing

Ageing is a process that involves a decrease in skin structural proteins (elastin, collagen,

hyaluronic acid) synthesis leading to loss in skin elasticity, laxity, integrity and finally gives rise to

visible signs of ageing. Apart from decreased protein synthesis, age-dependent down-regulation of

skin elasticity is also contributed by an up-regulation of the corresponding proteinases. Hence, the

most appropriate anti-ageing strategy in order to attain smooth and healthy skin is by controlling the

skin structural constituents’ degradation via proteinase activity regulation. In addition, frequent and

repetitive exposure to ultraviolet (UV) results in over-production of reactive oxygen species (ROS)

that induces oxidative cellular stress and promotes genetic alterations thereby affecting matrix

protein structure as well as functions, which eventually results in skin damage. In order to overcome

this issue, there is a strong need to scavenge these free radicals [144].

β-carotene, is one of the microalgae pigments commonly synthesized by D. salina that assists in

the prevention of free radicals that initiate premature ageing [143,148,149]. Another carotenoid

compound, lutein is also reported for its protective reactions towards UV radiation and can be

obtained from several microalgae (Scenedesmus salina, Chlorella, C. vulgaris, Scenedesmus obliquss, D.

salina and Mougeotia sp. [143,150]. Lycopene is also a microalgae carotenoid pigment that is employed

in skincare products for its anti-ageing properties where this compound neutralizes oxygen-derived

free radicals [143,145,151].

5.2. Antioxidant/Stress protection

Oxidative stress process impacts the skin mainly in terms of premature ageing, uneven skin

tone/texture and also might disintegrate the essential proteins. Once the collagen and elastin in the

dermal skin layer are diminished via oxidative stress, this also involves significant DNA damage,

inflammatory response, reduced antioxidant protection and the generation of matrix metalloprotein.

In the long run, this results in expedited ageing process along with significant appearance of wrinkles

with loss of elasticity [152]. Fucoxanthin, that is predominantly present in Phaeodactylum tricornotum,

Odontella aurita and Isochrysis aff. Galbana is one important metabolite that was identified to portray

antioxidant activity as well as preventing oxidative stress. Astaxanthin is reported to possess higher

antioxidant activity compared to vitamin A or E, and a recent study by Davinelli and co-workers in

the year 2018 highlighted its potential as a potent anti-wrinkle and antioxidant agent [153].

5.3. Free radical/Sunscreen protection

UV radiation is reported to be beneficial in limited duration; however, prolonged exposure is

not advisable as this will result in severe skin damage. In order to prevent these harmful effects,

various skincare products are usually applied especially by women that could range from sunscreens,

sunblock lotions or anti-ageing serum. Microalgae-derived carotenoid pigments such as astaxanthin,

lutein, zeaxanthin and canthaxanthin found abundantly within Dunaliella and Haemotococcus sp. are

noted for their protective properties against extensive sun damages [154]. In addition, orange-

pigmented violaxanthin compound isolated from Nannochloropsis oceania is proven to significantly

block UVB-detrimental effects along with decreased cell viability and increased ROS production

[155]. Fucoxanthin, is another microalgae-derived pigment that has been shown to impart protective

effect against sunburn [156].

5.4. Pigmenting agent

Almost all cosmetic products are incorporated with synthetic colourants which attracts much

attention of the cosmetic industry towards identifying colourants from natural sources for long-term

sustainability. Microalgae pigments fit well into these criteria where it is classified as a naturally

sustainable source with added health benefits that include UV protection, anti-ageing, antioxidant

and anti-bacterial properties. Carotenoids, chlorophylls and phycobiliproteins encompass the major

classes of pigments in microalgae; where they impart various colours ranging from green, yellow,



21

brown and red making it suitable as an alternative to synthetic colourants [157]. Chlorophylls are

extensively applied for use as cosmetic colourants, along with its other use in deodorants as an odour-

masking agent. Chl a, predominantly found in Chlorella and Spirulina sp. is a blue-green compound

and widely used for its stable nature [145]. In contrast to chlorophylls, astaxanthin gives rise to strong

red pigmentation, that are highly useful in most cosmetic products [158]. Phycoerythrin exerts intense

pink fluorescence and is formulated for use in lipsticks, eye shadow, and other make-up essentials.

Phycocyanin, in contrast, imparts blue fluorescence and is sourced from cyanobacteria (Spirulina sp.

or Arthrospira sp.). In the Japanese cosmetic market, phycocyanin isolated from Arthrospira sp. has

been commercialized as cosmetic colourants and eye shadow products [143].

Apart from being used as colourants, these microalgae-pigments are also utilised in tanning

pills. Tanning, is a process defined as the skin adaptation to UV exposure whereby the increased

melanin level plays role in protecting the skin from sunlight rays that induce free radical formation

[143,159]. Cantaxanthin, a pigment that is found predominantly in green microalgae (Chlorella sp.,

Haemotococcus sp. Nannochloropsis sp.) is one of the most common ingredients utilised in tanning pills.

Canthaxanthin imparts its tanning effect by darkening the skin via deposition of its red-orange colour

in the epidermis and subcutaneous fat. However, it is important to address that canthaxanthin

tanning pills are yet to be approved by FDA where adverse effects of urticarial, hepatitis and fatal

aplastic anemia have been noticed with daily consumption of these pills [143].

5.5. Whitening agent

In contrast to tanning effect, the favourability towards fairer skin tone especially among Asian

women has attracted much interest on whitening products as part of their beauty regime. Whitening

effect of skin arises with the inhibition of tyrosinase enzyme, which plays vital role in melanin

biosynthesis. Tyrosinase catalyses melanin synthesis via L-tyrosine hydroxylation to 3,4-dihydroxy-

L-phenylalanine (L-DOPA) as well as oxidation of L-DOPA to dopaquinone followed by further

conversion to melanin [160]. Several microalgal species are noted for their superior tyrosinase

inhibition activity, such as N. oculate or H. pluvalis. Both zeaxanthin and astaxanthin pigments from

these microalgae species are addressed for their anti-tyrosinase ability, making them suitable for

cosmetic products intended for skin whitening [143,161,162].

5.6. Moisturising agent

Moisturisation is a very critical step in preventing premature ageing of the skin, by maintaining

its elasticity and radiance. The level of moisture retained within the human body is correlated with

regulated water transport, corneocytes and hyaluronic acid as well as washing frequency [163.164].

In common practice, cosmetic range of products intended for moisturising effect is formulated with

hydroxy acid however; this elevates the price range due to its limited supply. As such, microalgae-

derived polysaccharides are preferred for its abundance as well as environmental-friendly nature. A

review by Wang and colleagues highlighted the potential of an extract from Chlorella vulgaris that

functions to support skin tissue along with collagen synthesis that assists in reducing wrinkle

formation [164].

5.7. Anti-inflammatory

Another vital and challenging issue of cosmetic products lies on the effect of neurogenic

inflammation that imparts its implications of irritation and itching as the adverse effects. It is

worthwhile to understand that most skin metabolic processes are affected by numerous signals in

response to stress-induced brain nervous stimulus [165]. These signals include secretion of different

hormones/substances from the various skin cells, such as indicated in Table 4 below.



22

Table 4. Anti-inflammatory secreted signals of the different skin layers.

Skin cells Secreted compounds Reference

Keratinocytes, melanocytes Corticotropin-releasing hormone (CRH), adrenocorticotropic hormone (ACTH), catecholamines

[165-167]

Dermal fibroblasts ACTH, cortisol, prolactin

Skin nerve endings Adrenaline, noradrenaline, substance P

Sebaceous glands CRH, prolactin

Cutaneous nerve endings and almost all skin cells

Produces and responds to special cytokines (neurotrophins) [165,168,169]

In response to nervous stress, the neurotrophins signal becomes crucial for some inflammatory

process (atopic dermatitis, psoriasis) where it induces proliferation of cutaneous nerve endings along

with symptoms ranging from itchiness and pain [165,170,171]. Despite of the limited findings

available on microalgae extracts implications on skin disorders, however, there are several bioactive

compounds derived from microalgae that are shown to be effective. One such example is of the

compound astaxanthin where it is vital in suppressing neuropathic pain in rats; where this involves

inhibition of N-methyl-D-aspartate receptors that is also part of pain mechanism caused by

neurogenic inflammation [165,172,173].

5.8. Industrial applications of microalgae in cosmeceuticals

Microalgae-based products have gained much popularity, and are continuously ventured.

Although the marketed products keep increasing over the years, yet it is still under par compared to

its active ingredient potential. Most of the products are those focused mainly on antioxidant and

photo-ageing protections; where there are still much more to be explored in terms of products

intended for skin appendages treatment, modulation of fat adipokines as well as the skin microbiota

[165]. At the same time, it is still important to highlight that it is economically disadvantageous in

microalgae exploitation as an isolated active compound source due to the relevantly high cost of

biomass production as well as purification [3,165,174,175]. Despite of these, continual

recommendation and exploration to utilise microalgae-derived metabolites for the synthesis of

products with multiple uses are inter-related with it being fully natural source along with its

numerous benefits especially in cosmetic care [165,176].

In a review by Mourelle and colleagues, it is highlighted that several cosmetic-based industries

have invested in establishing their own microalgae cultivation. These include renowned companies

such as Louis Vuitton Moët Hennessy (Paris, France), Danial Jouvance (Carnac, France) and AGI

Dermatics (America). One of the latest highlighted products in 2020 is the Argan Beta-Retinoid Pink

Algae Serum launched by Josie Maran. This product is comprised of the main compound β-carotene

extracted from pink algae that play vital roles to eliminate fine lines, wrinkles, dark spots, dullness,

dry and flaky skin. The serum is also enriched with organic argan oil, quercetin and is claimed to be

free of synthetic colours, fragrance, parabens, petrochemical or phthalates [177]. In addition, various

other microalgae-rich extract products are also commercialized on a continual basis. Among these,

several products available in the market are as shown in Table 5 below.



23

Table 5. Selected representation of the commercialized microalgae-derived cosmetic care products. 1

Microalgae Commercial name/Company Application Reference

Dunaliella salina Blue RetinolTM Stimulates skin cell growth and proliferation [178]

Nannochloropsis oculata Pentapharm Promotes excellent skin-tightening [145,178]

Chlorella vulgaris Dermochlorella/CODIF Recherche and Nature Stimulates collagen synthesis, supports tissue regeneration, reduces wrinkle

formation, anti-ageing

[3,179]

Arthospira sp. Protulines, Exsymol Assists in skin-tightening and repairs signs of ageing skin [147]

Phaeodactylum tricornutum Givaudan Prevents premature ageing [145]

Argan Beta-Retinoid Pink Algae Serum Josie Maran Eliminate fine lines, wrinkles, dark spots, dullness, dry and flaky skin [177]

Porphyridium sp. Alguard®/Frutarom Anti-wrinkle, protection from UV damage [154,180]

Porphyridium cruentum SILIDINE®/Greentech Improves skin aspect, decrease redness, vascular toning [145]

Cicatrol®/Greensea Anti-ageing, antioxidant [181]

Tetraselmis suecica O+ Gold Microalgae Extract®/Greensea Anti-inflammatory [181]

Scenedesmus rubescens Pepha®-Age/DSM Nutritional Products Protects from photo-ageing, pro-collagen [182]

Nannochloropsis oculata Pepha®-Tight/DSM Nutritional Products (Pentapharm) Protects from oxidative stress, simulates collagen synthesis and skin

tightening

[183]

Dunaliella salina Pepha®-Ctive/DSM Nutritional Products (Pentapharm) Stimulates collagen synthesis, cell proliferation, energy metabolism

Dunaliella salina, Haematococcus pluvialis REVEAL Color Correcting Eye Serum Brightener/Algenist Treats uneven and dull skin, dark circles under eye, gives brighter complexion [184]

Chlorella vulgaris Phytomer/Phytomer Protects skin, neutralises inflammation [154]

Chlorella sp. Golden ChlorellaTM/Terravia Holdings Hydrates skin and hair [145]

2



24

Apart from the commercialized products, a recent study in 2017 reported on the potential of a

novel, non-fastidious freshwater microalgae (Chlorella emersonii KJ725233) for possible application in

cosmeceuticals accounted by its anti-ageing, antioxidant and anti-inflammatory properties. Its ability

to inhibit elastase, hyaluronidase, collagenase and to scavenge free radicals supports skin tissue that

gives rise to skin rejuvenation. In addition, phytol compounds, shown to reduce the proteinases

activity and thus, reduces the dermis inflammation [144].

6. MICROALGAE IN BIOFUELS AND ENERGY

Energy production has become the priority of the world’s population due to the fuel demand

for transportation, electric power generation and operation of manufacturing plants. There are two

groups of global energy sources; non-renewable (fossil) and renewable. The renewable energy

sources which have been investigated to displace the depleting fossil energy sources include solar,

wind, nuclear, geothermal, hydrogen, waves, tidal and algal biomass energy [185,186].

The first generation of biofuels comes from terrestrial crops (e.g. maize, sugarcane, sugar beet

and rapeseed) which has many drawbacks/limitations causing destruction of forests and the

abundant usage of water. The second generation is from forest residues, lignocellulosic agriculture

and from non-food crop feedstocks which disadvantage is the land use [187,188]. Biofuels are the

renewable energy sources produced from algal biomass which is the third generation of biofuels and

is a promising alternative method to the previous methods. The advantages of using algae to generate

fuels/energy are that it can be produced all-year-round, easy to cultivate in water without much

effort, requires less water compared to terrestrial crops, does not require pesticides or herbicides thus

reducing the cultivation cost [189,190].

Alga biofuel has the potential to meet global demand since they do not compete with the

production of food products. They can be exploited to biofuels inclusive of biodiesel,

bioethanol/biobutanol, syngas, biochar, biogas and energy (electricity/heat) through various

processing technologies such as thermochemical conversion (transesterification, thermochemical

liquefaction, direct combustion, gasification and pyrolysis) and biochemical conversion

(fermentation, anaerobic digestion and photobiological hydrogen production) (Figure 6) [16,191].



25

1

2 3

Figure 6: Various methods of producing biofuels/energy from microalgae biomass. Adapted from [16,191]4

Microalgae Biomass

BiochemicalConversion

Transesterifi-cation

Biodiesel

Fermentation

Bioethanol/

Bobutanol

Anaerobic Digestion

Biogas/

Methane

Biophotolysis

Hydrogen

Thermochemical Conversion

Gasification

Syngas

Thermochemical

Liquefaction

Bio-Oil

Pyrolysis

Bio-Oil

Syngas

Charcoal

Direct Combustion

Energy

Electricity

Heat



26

6.1. Biochemical Conversion

Biochemical conversion is the conversion of microalgae biomass to biofuels with the aid of

biocatalysts, such as enzymes and microorganisms such as bacteria and yeast [192]. This method

works well with high-water content biomass. The biochemical conversion includes

transesterification, fermentation and anaerobic digestion.

i)6.1.1 Transesterification- Biodiesel

Fatty acid methyl ester (FAME) called as biodiesel is produced by transesterification or

esterification; a chemical reaction between alcohol and triacylglycerides without or with catalysts

such as alkaline catalyst (sodium hydroxide or potassium hydroxide) or acid catalyst (sulphuric,

sulphonic, phosphoric, and hydrochloric acids) [193,194]. Many species of microalgae such as

Scenedesmus obliquus, Neochloris oleabundans, Nannochloropsis sp. and C. vulgaris contain a high content

of lipids including triacylglycerides that are needed for the biodiesel production [195,196]. The

production of biodiesel involves several stages including lipid extraction using solvents; methyl or

ethyl alcohol (unrefined lipid), followed by lipid recovery with centrifugation-solvent evaporation

(purified lipid and free fatty acids), transesterification or esterification (biodiesel production). The

final step of this process is the removal of the solvent for the biodiesel recovery [196]. Biodiesel from

renewable resources such as microalgae has several advantages compared to petroleum diesel; non-

toxic, biodegradable, renewable and contains low levels of carbon monoxide, soot, hydrocarbons and

particulates. It also discharges a low level of CO2 of up to 78% lower [197]. Many researchers have

look into the possibility of producing biodiesel alongside with bioethanol from algae biomass. A

study by Wang et al. [198] had shown encouraging results of Tribonema sp. with enriched lipid and

carbohydrate content for biodiesel and bioethanol production. The conversion rate of lipid extracted

with hexane-ethanol to biodiesel was 98.4% and the maximum yield of ethanol using yeast

Saccharomyces cerevisiae was 56.1%.

ii)6.1.2 Fermentation- Bioethanol

The chemical and enzymatic pre-treatment method are usually used in the production of

bioethanol. The common strong acids used include hydrochloric, sulfuric and nitric acid, whereas

enzymes such as amylases, cellulases and invertases are used. However, using enzymes can be rather

expensive if involves large scale production [199]. Sometimes the cell walls are disrupted using a

mechanical method such as by 24/24 freezing/defrosting cycle to release the intracellular substances

[200]. The fermentation of the treated biomass is usually using yeast Saccharomyces cerevisiae or

bacteria at warm temperature (30-40 oC) to obtain ethanol and a bioreactor is later used for up-scaling

[201,202]. The quality and quantity of the produced bioethanol are fully determined by the method

used and fermentation process parameters inclusive of pH, temperature and the fermenter organism

used [203].

Previous studies have shown that several microalgae species can act as an effective feedstock for

the production of bioethanol. The species include Chlorococcum humicola, Scenedesmus abundans PKU

AC 12, Chlorella vulgaris FSP-E, Scenedesmus obliquus CNW-N, Porphyridium cruentum, Desmodesmus

sp, Spirulina platensis and Scenedesmus acuminatus (Refer Table 6). A comparison study of red

microalgae (P. cruentum) culture conditions for bioethanol production was done by Kim and co-

workers in 2017 [204]. The results indicated ethanol conversion yields from P. cruentum cultured in

freshwater was much higher (70.3%) than the P. cruentum cultured in seawater (65.4%). Recently,

Chandra et al. [205] study the effect of the cultural variables on the carbohydrate accumulation of

Scenedesmus acuminatus to produce bioethanol. The bioethanol yield obtained was 0.12 g/g with the

supplementation of lysine in the medium culture and at higher initial culture pH (pH 9.0).



27

Table 6. Bioethanol production from various microalgae species.

Microalgae Ethanol yield (g ethanol/g substrate) Reference

Chlorococcum infusionum 0.26 [206]

Chlamydomonas reinhardtii 0.24 [207]

Chlorococcum humicola 0.52 [208]

Scenedesmus abundans PKU AC 12 0.103 [209]

Chlorella vulgaris FSP-E 11.66 (87.59%) [210]

Scenedesmus obliquus CNW-N 8.55 (99.8%) [211]

Desmodesmus sp. FG

strain SP2-3 (unidentified green microalga)

0.24 [212]

Scenedesmus acuminatus 0.12 [205]

iii)6.1.3 Anaerobic Digestion- Biogas/methane

Microalgae can be converted to biogas through anaerobic digestion which is a biochemical

process that mineralizes organic material through the action of microorganisms in the absence of

oxygen [213]. Biogas is mainly made up of a mixture of methane, CH4 (50-70%), CO2 (30-45%) and

traces of other gases such as hydrogen, H2 (<2%) and hydrogen sulphide, H2S (<3.5%). The process

involves three stages; hydrolysis, acetogenesis/acidogenesis and methanogenesis [214]. Biogas

production is often reduced due to the thick and rigid microalgae cell wall, therefore it is normally

incorporated with production of other bioproducts such as biodiesel or bioethanol [215]. To

overcome the cell wall problems, pre-treatment is usually done to enhance the process. Pre-treatment

with heat was found to enhance the production of biomethane from Chlorella sp. by 11% as the

temperature was increased to 90°C from 70°C for 0.5h compared to the control [216].

iv)6.1.4 Biophotolysis- Hydrogen

Green microalgae and cyanobacteria can produce hydrogen through biophotolysis process.

They possess chlorophyll a and the photosynthetic systems; Photosystem II (PS II) and Photosystem

I (PS I), that enables them to perform photosynthesis by absorbing solar energy and converting water

into hydrogen and oxygen [217,218]. Biophotolysis can be divided into two; direct and indirect

pathways. During direct biophotolysis, water splitting at PS II generated electron and proton, giving

rise to H2 in both green algae and cyanobacteria. For indirect biophotolysis, the degradation of carbon

compounds will generate protons and electrons for the production of hydrogen which are mostly

found in cyanobacteria [219].

There are several green microalgae and cyanobacteria such as Tetraspora sp. CU2551, R. rubrum,

R. spheroides, Rhodobacter capsulatus, C. vulgaris, C. reinhardtii, Anabaena sp. and Nostoc sp. which have

been widely studied for the hydrogen production [220-223]. The model microalga that is usually used

for research is the C. reinhardtii since some of its organelles such as mitochondrial, nuclear genomes

and chloroplast have been successfully sequenced [224].

6.2. Thermochemical Conversion

Thermochemical conversion is a process whereby microalgae biomass is converted to biofuels

with the involvement of heat at a different temperature depending on the final product desired. This

method can be used for both dry and wet biomass. Normally, in the thermochemical conversion no

chemicals are added and the period for producing biofuels from this method is shorter than the

biochemical conversion [225]. The thermochemical conversion methods include gasification,

thermochemical liquefaction, pyrolysis and direct combustion.

6.2.1 Gasification- Syngas



28

Syngas or synthesis gas is mainly composed of methane, hydrogen, carbon monoxide and

carbon dioxide that can be used to generate heat or electricity [185,226]. Microalgae biomass is

converted to Syngas through a gasification process which utilises partial oxidation with a mixture of

oxygen and steam at high temperatures (>700°C) [227].

6.2.2 Thermochemical Liquefaction- Bio-Oil

Crude bio-oil is produced by thermochemical liquefaction in the presence of a catalyst at a

temperature between 300-350°C [228]. Hydrothermal liquefaction is the preferred method for wet

algae biomass which uses water and elevated pressure [227]. The thermochemical liquefaction yield

of microalgae depends on various parameters including type of catalysts and reaction temperature

[229].

6.2.3 Pyrolysis- Bio-Oil, Syngas, Charcoal

Bio-oil, syngas and charcoal can be produced through pyrolysis; the conversion of microalgae

biomass in the absence of oxygen at medium to high temperatures (400–600°C) [230]. Different

products produced at different stages of temperature and duration of the process. Bio-oil is produced

with a moderate temperature (500oC) and a short-time exposure to vapour known as flash pyrolysis.

Syngas is produced through fast pyrolysis at moderate temperature (500oC) and moderate time

exposure to vapour. Charcoal is produced through slow pyrolysis at a lower temperature (400oC) and

long-time exposure to vapour [231].

6.2.4 Direct Combustion- Energy (Heat/Electricity)

Direct combustion is the burning of the microalgae biomass to generate heat, energy or

electricity. It is normally done in a boiler or steam turbine at a temperature above 800oC in the

presence of oxygen. The process can produce 300 MW for domestic or a large-scale industrial process

[16]. However, the overall operation cost may increase due to the pre-treatment requires by the

microalgae biomass such as drying [232].

6.3. Industrial applications of microalgae in biofuels

The potential of microalgae to be used as a source of biofuel in the future to replace the fossil

fuel has always been a concerned [233]. However, there are several challenges that need to be

addressed to enable success which include the development of low-cost, effective cultivation systems,

efficient and energy-saving harvesting techniques because there are problems to discard large

volumes of water during harvesting and methods for oil extraction and conversion that are

environmentally friendly and cost-effective by selecting the best strain for higher production of

biofuel [186,234]. In terms of cost, the harvesting process may involve a lot of money and

commercialization will be too expensive as reported by the U.S. Department of Energy (DOE) who

has been doing research in this area for about 16 years from the year 1980s to 1990s [235]. The only

oil major company known to be still investing in the microalgae biofuel project is the ExxonMobil. In

2017, after 8 years of collaboration with a biotechnology company Synthetic Genomics, they

announced a major breakthrough in producing microalgae with 40 percent more lipids while

maintaining growth rates using CRISPRCas9 genome editing technique [236]. However, to date no

recent developments are known regarding this issue. Although the microalgae-based biofuel

production could not be commercialised in near future, there is still a possibility of using microalgae

for biofuels in the future because of the abundance of resources such as the significant amount of

land, water and CO2 available to support the algal biofuel technology [235]. Lately, algal biofuel

production has attracted interest again from many researchers to explore in this field to be

commercialised later. The areas of study and development that need to be considered to achieve



29

success are in the cultivation, harvesting and extraction methods where the growth rate of microalgae

need to be enhanced with a more robust alga growing systems, new species/strains with high lipid

content need to be sought or the lipid content in the microalgae needs to be enhanced in various ways

which are found to be appropriate and the development of an efficient method for the biofuel

extraction needs to be explored so as not to increase the production cost [235].

7. MICROALGAE IN BIOFERTILISERS

Biofertilisers are natural substances or products containing live microorganisms that enhance

the chemical and biological properties of the soils, revive soil fertility and stimulate plants growth

[237]. Plants need nitrogen to grow and the lack of such component can be overcome by giving

fertiliser at an adequate rate. However, excessive and prolonged use of chemical or synthetic

fertilisers resulted in environmental pollution that will eventually cause an imbalance in the

ecosystem [238]. As an alternative, microalgae have been extensively studied to see its potential as

plant biofertilisers and also biostimulants.

The majority of cyanobacteria can fix nitrogen from the atmosphere and several species are

known to be efficient as cyanobacterial-based biofertilisers such as Anabaena sp, Nostoc sp. and

Oscillatoria angustissima [239-241]. Some of the various green microalgae and cyanobacteria species

successfully used as biofertilisers to enhance crops growth include Acutodesmus dimorphus, S.

platensis, C. vulgaris, Scenedesmus dimorphus, Anabaena azolla and Nostoc sp. (Table 7), with Chlorella

vulgaris as one of the most commonly used microalgae in biofertiliser studies. The germination of

Hibiscus esculentus was accelerated using combined seed and soil treated with C. vulgaris. Significant

improvement of the soil nutrient content and microorganism count before treatment with the

biofertiliser was also observed [242].

Table 7. Microalgae species and their use as plant biofertiliser and biostimulant.

Microalgae Crops Reference

Acutodesmus dimorphus Tomato [243]

Spirulina platensis,

Chlorella vulgaris

Maize [244]

Chlorella vulgaris Hibiscus esculentus [242]

Chlorella vulgaris (UTEX 2714),

Scenedesmus dimorphus (UTEX 1237)

Rice [245]

Spirulina platensis,

Chlorella vulgaris

Onion [246]

Chlorella vulgaris Tomato and Cucumber seeds

[247]

Scenedesmus sp. Rice [248]

Anabaena azolla Rice [249]

Nostoc muscorum

Nostoc rivulare

Maize [250]

The treatment using green microalgae/cyanobacteria have shown many beneficial effects on the

plants and soils. The seed germination, plant growth, yield and the nutritional value of the crops is

enhanced besides the improvement of the soil fertility. The carbon and organic content of the soil

were accelerated due to the excretion of carbon (exopolysaccharides) by the green

microalgae/cyanobacteria into the soil and the degradation of the biomass and grazing activity add

on to the increment [251-254]. Studies had shown that those factors influence the microbial activity



30

and biomass of other microflora and fauna in the soil which eventually will stimulate the growth of

the crops [251,255].

A study by Bumandalai et al., [247] exhibited the potential of Chlorella vulgaris as biofertiliser for

the germination of tomato and cucumber seeds. The length of the tomato and cucumber roots and

shoots were improved using algal suspensions of 0.17 and 0.25 g/L, respectively. Treatment of plants

with A. dimorphus biofertiliser before seedling transplant showed enhanced germination, increased

production of branches and flowers compared to the control group and the treatment group applied

during transplant [243]. Nayak et al., [248] successfully used de-oiled microalgal biomass of

Scenedesmus sp. as biofertiliser to improve the growth of rice plant.

The cyanobacteria can colonize different parts of a plant tissue such as in the roots and shoots,

stimulating the nitrogen fixation and phosphorus solubilising microbial population on that parts,

thus enhancing and improving the growth, nutritional status and defence mechanism of the plant

and soil fertility [256-261]. Apart from that, cyanobacteria also produce siderophores (organic

compounds) to facilitate in chelating micronutrients (e.g Fe and Cu), known as biomineralization to

make them easily accessible for plants growth [262,263].

Many green microalgae/cyanobacteria are also reported to excrete intracellular hormones to

their surroundings which help to stimulate plants growth [264-266]. Examples are the green

microalgae, Chlorophyta and Cyanophyta [267] and some cyanobacteria strains which produce

cytokinins and auxins which help to stimulate plants growth parameters such as shoot length, root

length, spike length and weight of seeds [268].

The use of green microalgae/cyanobacteria as biofertiliser can increase the activity of defence

mechanism of the plants and improve their immunity by increasing the plant RNA activity,

producing nutrient assimilating enzymes; dehydrogenase, nitrate reductase, acid or alkaline

phosphatase, generating antioxidant and plants defence enzymes such as peroxidase, polyphenol

oxidase, phenylalanine ammonia lyase and β-1, 3-endoglucanase in root and shoot of the plants

[259,269,270]. Different strains of microalgae might provide different levels of defence mechanisms

as shown by a study by Babu et al. [271]. Three different cyanobacteria (Anabaena laxa RPAN8,

Calothrix sp. and Anabaena sp. CW2) inoculated in wheat plant showed the highest activity of

peroxidase, polyphenol oxidase and phenylalanine ammonia-lyase with Calothrix sp. The results

could suggest that using a combination of different types of microalgae with different abilities as

biofertiliser, may contribute to the increase of plants immunity.

Some researchers had tried using a mixture of different microalgae species or a combination of

microalgae and other organic or chemical fertilisers to further enhance its effectiveness. Maize plant

treated with C. vulgaris and S. platensis along with cow dung manure for 75 days under greenhouse

conditions, appeared to improve the growth and yield of the maize plant [244]. Jochum et al., [245]

utilised a mixed culture of C. vulgaris (UTEX 2714) and Scenedesmus dimorphus (UTEX 1237) biomass

as biofertiliser for rice plant. The biofertiliser showed efficacy with a significant increase in height of

the rice plant. Growth parameters of onion plants were found to enhance with the application of a

mixture of S. platensis and C. vulgaris showing higher growth rate and yield compared to the control

group [246]. Consortia of green microalgae and cyanobacteria also showed promising results with

the improved activity of soil microbial, increase in soil organic carbon, macro- and micronutrients

and enhanced plant growth and yield [272,273]. Figure 8 summarises the function of microalgae

biomass as biofertilisers.

7.1. Industrial applications of microalgae biofertiliser

The cost of using chemical fertilisers are increasing every year due to the high demand for foods

from plants since it is one of the major important components for plant growth, especially for food

production. Microalgae-derived biofertiliser can be an alternative for chemical fertiliser and has

gained more popularity due to the natural effects they provide to the crops and the surrounding soil.

The chemical fertiliser may cause several problems, among them are the pollution (soil, water, air) to

the environment, reducing input efficiency, decreasing food quality, developing resistance in weeds,



31

diseases and insects, micronutrient deficiency in the soil, degradation of soil and causing toxicity

effect to different beneficial organisms living in the soil [274]. On the other hand, the biofertilisers are

eco-friendly, more economical and more efficient [238]. Several things that are considered before the

commercialisation of microalgae as biofertiliser are the formulation of the inoculate, the nature

application of the products, packaging issues and also products storage [275]. Several countries have

successfully commercialised the microalgae-derived biofertiliser products, which include the

developing and developed countries. Previously in 2019, a Spanish company, Biorizon Biotech, S.L.

had applied a patent for the method of obtaining concentrates of biofertilisers and biostimulants for

agricultural use from green microalgae and cyanobacteria biomass [276]. Among the available

microalgae-derived biofertiliser products in the market are as shown in Table 8.

Table 8. Commercialised microalgae-derived biofertiliser

Microalgae Commercial name Company Country Reference

Cyanobacteria

(Blue-green algae)

Skipper Khad® and

Power Play-90

WSG

Ecological Products

Industries

India [277]

Spirulina sp. Shwe Awzar®

June Industry Limited

Myanmar [278]

Spirulina sp. Algafert® Biorizon Biotech

Spain [279]

Chlorella vulgaris Terradoc® Mikroalg Food and

Agriculture

Industries

Turkey [280]

Cyanobacteria TerraSyncTM

Accelergy Corporation USA [281]

Chlorella sp. EMEK MCT Tarim Ltd Turkey [282]



32

1



33

8. MICROALGAE IN WASTEWATER TREATMENT

Microalgae can utilise nitrogen, phosphorus, carbon and chemicals such as heavy metals from

waste water as their source of nutrients and this is called as phycoremediation [283]. Conventional

wastewater treatment plants (WWTPs) is designed basically to remove organic matter and nutrients.

Nitrogen (N) and Phosphorus (P) are the most important pollutants in the aquatic environments

because they are discharged into water bodies in huge quantities. Removal of N and P using

microalgal remediation technology has high removal efficiencies which N and P contaminants can

be completely removed from WWTPs, lower costs operation, zero sludge generation and high-value

products can be generated such as fatty acids, pigment, biomass, biodiesel and unicellular protein

[284-287].

New technology in removing heavy metals such as zinc, copper, lead, mercury, chromium and

cadmium from the wastewater (WW) is by using microalgae biomass. Microalgae biomass is an

option owing to low obtaining costs and close to 100% efficiency in the removal of heavy metals

[288,289]. Microalgae also able to remove colorant such as aniline used by textile, cosmetics and food

industries. A major portion of colorant removal is due to the interaction of the molecule with the

reactive sites of the cell wall which permits the elimination of the colorant in a rapid manner [290,291].

Emerging contaminants (ECs) are molecules produced in industries such as pharmaceutical

(used for human beings and livestock) and chemicals, including compounds such as hormones,

antibiotics, plasticizers, antipyretics, antifungal and surfactants which cannot efficiently remove by

WWTPs [292,293]. The removal of ECs is normally done by physicochemical processes such as photo-

oxidation, ozonisation, or oxidation via metallic catalysis. Algae-based bioreactors have gained

special research interest as a promising way to remove pharmaceuticals-based ECs from the

wastewater either partially or completely. A wide spectrum of technologies has been used for the

removal processes for ECs, mainly grouped as physical adsorption, chemical advanced oxidation and

biological degradation processes and combination. Laboratory scale (open pond and bubble column

photobioreactor) showed high removal percentages have been reached for ciprofloxacin, metoprolol,

triclosan, and salicylic acid (>90%); moderate removal for tramadol and carbamazepine (50-90%), and

trimethoprim and ciprofloxacin show fewer promising results with very low removal (<10%) [294].

Based on the latest research, phenol and its derivatives could be metabolized and degraded by

algae. The ability of algae to remove phenolic compounds dependant with the type of algae or

substrate, degradation time and initial pH. However, algae have a certain tolerance range to phenol

[295,296]. Antibiotics are other pollutants that have been released to the environments and create

antibiotics resistance. Recently, microalgae-based technology has been explored as a great potential

alternative for the treatment of wastewater containing antibiotics by adsorption, accumulation,

biodegradation, photodegradation, and hydrolysis [297]. Two freshwater algae species Scenedesmus

obliquus and C. pyrenoidosa were shown to have the capability to transform steroids such as

progesterone and norgestrel [298].

A device called Microalgae-microbial fuel cell (m-MFC) integrated process has been developed

to overcome the problem of fossil-fuel depletion and environmental pollution by generating electrical

energy from wastewater and sunlight, wastewater treatment, CO2 sequestration and biomass

production in single, self-sustainable technology [299,300]. Table 9 showed microalgae used in the

remediation of the pollutants present in the wastewater.



34

Table 9. Microalgae used in the remediation of the pollutants present in the wastewater.

Pollutants Microalgae bioremediation References

Nitrogen &

Phosporus

Phormidium sp, Spirulina maxima, Chlorella vulgaris, Scenedesmus dimorphus, Scenedesmus quadricauda, Chlorella sorokiniana, Chlorella vulgaris ESP-6 [284-287]

Heavy Metals Chlorella sp., Tetradesmus sp., Scenedesmus sp., Porphyridium sp., Chaetoceros sp., Oscillatoria sp., Sprogyra sp., Scenedesmus sp., Anacytis sp., Chlamydomonas sp.,

Anabaena sp., Ceratium sp., Scenedesmus sp., Calothrix sp., Arthrospira platensis

[288,301,302]

Colorants

Chlorella vulgaris, Cosmarium sp., Nostoc linckia, Spirogyra sp. [303-306]

Emerging Pollutants

Antibiotics

Phenol

Nannochloris sp., Mixture of algae-bacteria consortia in pilot high rate algae pond (HRAP), Mixture of algae-bacteria consortia (dominated by Coelastrum sp.) in HRAP,

Mixture of algae-bacteria consortia in 1-L HRAP S. obliquus, C. vulgaris, Chlorella sp., Scenedesmus sp., Chlamydomonas mexicana, Chlorella sorokiniana

Chlorella vulgaris

Chlorella pyrenoidosa

[307-311]

[296,312]



35

9. MICROALGAE IN FEED

Microalgae biomass have also been successfully employed in feed formulations for different

animals like cattle, fish, goat, lamb, poultry, pigs and rabbits. In aquaculture, species of genus such

as Nannochloropsis, Isochrysis, Pavlova, Phaeodactylum, Chaetoceros, Skelotenma, Thalassiosira and

Tetraselmis are used as biomass [313]. A microalgae, D. salina has been applied in animal feed for its

rich protein (57% d.w.) and carbohydrates (32% d.w.) contents [314]. Microalgal based feed affects

the animal's physiology as well as it improves its immune response and fertility. The combination of

5–15% of algal biomass, mixed with animal feed, can be used safely as a partial replacement for

conventional proteins. However, the use of higher concentrations of microalgae biomass results in

less feed intake by some animals due to the lower palatability of the feed. The quality of proteins with

microalgal sources have a comparable or superior functionality (either as foam, emulsifier,

solubilizer, surfactant, or gelling agent) equal or even greater than other commercial protein sources

[315]. Chlorella biomass showed that it can be digested easily up to 5% in the form of paste [44].

Microalgae can provide an important source of essential fatty acid to fish. DHA and EPA are not

synthesized by fish and the only resource to enrich them in fish are by accumulating them in proper

algal paste and store them as fish feed [316,317]. Microalgae, like C. vulgaris and A. platensis, consist

of amino acid profiles that show similarities to soybean which is currently the main source of protein

used in feed. In addition, the microalgal biomass is a potential source of minerals; C. vulgaris, A.

platensis, Micractinium reisseri, Nannochloris bacillaris, and Tetracystis sp. showed very high contents of

iron in contrast to soybean [318].

10. MICROALGAE IN PROTEOMICS

Genetic engineering has been used to manipulate microalgae which allow researchers deducing

metabolic pathways in order to construct new algal metabolism methods. This will enable to produce

new molecules useful for biotechnological applications [319]. Microalgae proteomics can give the

information of how genes of interest are being expressed as proteins which will help researchers to

find proteins with biotechnological applications such as anti-inflammatory proteins [320].

The potential of microalgae in the production of proteins with biotechnological application has

been explored. C. reinhardtii has been extensively studied for therapeutic protein production and

more than 100 such proteins have been expressed successfully in this system [321]. Microalgae-based

biomanufacturing is preferred due to effectiveness in terms of cost and energy, fewer contamination

chances, and simplified downstream processing. Malaria detection protein which are used in ELISA

testing and cell-traversal protein as an antigen from sporozoites and ookinetes of mosquitoes have

been successfully expressed and produced in C. reinhardtii chloroplast [322]. Phycobiliproteins from

cyanobacteria and red algae have been described to present antioxidant, hepato-protective, anti-

inflammatory, immune-modulating, and anticancer effects [51,323-326]. Chlorella pyrenoidosa and

Chlorella vulgaris have shown anti-hypertensive and anti-tumour activities from its isolated peptides

[325].

A protein isolated from Chlorella sorokiniana has high-value bioactive peptides with nutraceutical

and pharmaceutical application, using proteomics techniques [332]. Most of the proteomics

approaches in the area of microalgae research are focused in development of efficient biofuels

production [327-330].

11. CONCLUSION

Microalgae are promising source of generous amount of metabolites with essential health

benefits. Continual exploration for novel microalgae species along with isolation of its bioactive

compounds are in great demand for diverse applications in various fields and industries as raw

material, biomass or high-quality extracts. Despite of the excellent and superior benefits, there are

several limitations that should be addressed as well. The significantly high incurring cost of the

isolation might result in over-priced products, where it is believed this could be partly subsidised



36

with utilisation of single solvent to generate high extract yield. In addition, it is essentially important

to highlight that these are natural extracts with unique composition and varies subject to the season,

cultivation condition, as well as the extraction method. Studies are also focusing on extraction of

more than one product from a single biomass for a cost-efficient industry. On the other hand, the

process of bringing a drug candidate to the market requires extensive pre-clinical testing and clinical

trials to determine the safety and efficacy of the drug before it is approved by the FDA and is a very

costly and time-consuming process. Hence, not all potential bioactive will eventually reach the

market and consumers. Indeed, low yield of bioactive and strenuous and expensive purification steps

have been regarded as the main challenge in producing economically viable drugs from microalgae.

Therefore, to overcome these issues some of these active compounds are currently expressed in

suitable vectors to ease purification and hence reduce the purification and production costs. Efforts

on cost reduction by studying effect of different culturing conditions such as light intensity, nutrient

availability and harvesting time on the metabolite yield and bioactivity as well as producing high-

yield microalgae species via genetic modification approaches are currently pursued to accelerate the

commercialization process of some of these bioactives. Meanwhile, advancements in biotechnology

applications can help overcome some issues of poor oral bioavailability or instability in the

gastrointestinal tract of some microalgae bioactives like peptides and fatty acids. This includes novel

approaches using encapsulation technique and nano formulation to improve the solubility and

bioavailability of some microalgae derived compounds for treatment of diseases. Furthermore,

genetic engineering approaches expressing microalgae metabolites in natural organism for drug

delivery have been explored. Nevertheless, more studies especially in in-vivo models and clinical

trials are still needed to determine the safety and efficacy of these novel drugs prior to drug approval

and commercialization.

In conclusion, it is vital that more efforts should be taken in developing multi-functional range

of products, that are affordable and is inter-related with nutritional science that could cater for even

more health benefits comparatively to the ones in current market. These findings would bring much

more insights into the vast potentials of microalgae-derived metabolites that remain to be explored

and assessed. Further roadmap towards enhancing phycoeconomy is recommended by uplifting the

current biological and technology process by taking into account on sustainability and environmental

benefits.

Acknowledgments: The authors thank the Director-General of Health Malaysia and the Director of Institute for

Medical Research (IMR), National Institutes of Health, Ministry of Health Malaysia, for giving the permission to

publish this article. We also thank the staff of Nutrition, Metabolism and Cardiovascular Centre, Institute for

Medical Research, NIH for their continuous support.

Author contributions: Conceptualization: All authors.; Literature review: All authors.; Tables: All authors.,

Writing and review All authors.; Editing: All authors; Revisions and Final editing: All authors. All authors have

read and agreed to the published version of the manuscript.

Conflicts of Interest: The authors declare no conflict of interest.

List of Abbreviations

ACTH Adrenocorticotropic hormone

ALA α-linoleic acid

cGMP current Good Manufacturing Practice

CPC Chlorophytic polyculture

CRH Corticotropin-releasing hormone

CV-N Cyanovirin

DHA Docosahexaenoic acid

DOE U.S. Department of Energy

EAAI Essential amino acid index

ECs Emerging contaminants



37

EPA Eicosapentaenoic acid

EAAI Essential amino acid index

FDA Food and Drug Administration

FP-PBR Flat-plate open pond and closed photobioreactor

GRAS Generally Regarded as Safe

HRAP High rate algae pond

IC Inhibitory concentration

L-DOPA 3,4-dihydroxy-L-phenylalanine

m-MFC Microalgae-microbial fuel cell

PBR Open pond and closed photobioreactor

PUFAs Polyunsaturated fatty acids

ROS Reactive oxygen species

SVN Scytovirin

TPBR Tubular open pond and closed photobioreactor

TAG Triacylglycerides

TG Triglyceride

UV Ultraviolet

VCPBR Vertical column open pond and closed photobioreactor

WW Waste water

WWTPs Waste water treatment plants

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